Fusion pore formation and expansion induced by Ca2+ and synaptotagmin 1

Abstract

Fusion pore formation and expansion, crucial steps for neurotransmitter release and vesicle recycling in soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent vesicle fusion, have not been well studied in vitro due to the lack of a reliable content-mixing fusion assay. Using methods detecting the intervesicular mixing of small and large cargoes at a single-vesicle level, we found that the neuronal SNARE complexes have the capacity to drive membrane hemifusion. However, efficient fusion pore formation and expansion require synaptotagmin 1 and Ca(2+). Real-time measurements show that pore expansion detected by content mixing of large DNA cargoes occurs much slower than initial pore formation that transmits small cargoes. Slow pore expansion perhaps provides a time window for vesicles to escape the full collapse fusion pathway via alternative mechanisms such as kiss-and-run. The results also show that complexin 1 stimulates pore expansion significantly, which could put bias between two pathways of vesicle recycling.

Fig. 1.

(A) Lipid mixed fractions of docked vesicle pairs 1 min after docking. The first bar is with SNAREs only, and the second bar is with SNAREs and Syt1. The error bars represent SD. (B) A typical time trace of fluorescence dequenching of small sulforhodamine B molecules due to content mixing (9). More time traces for content mixing are shown in Fig. S6. The leakage, as opposed to content mixing, yields the fluorescence intensity near zero. Examples of photobleaching and leakage are shown in Fig. S7. The probability of the leakage of sulforhodamine B is ∼1%. The error bars, which represent SD, were obtained from three independent measurements. (C) Sulforhodamine B content mixing at various concentrations of SNAREs. No Syt1 is present. (D) Syt1/Ca²⁺ stimulate sulforhodamine B content mixing significantly. Total 192 time traces were used to generate the cumulative fusion time histogram.

Fig. 2.

Single-molecule content-mixing assay using large DNA cargoes. (A) Schematics of the assay. Vesicles reconstituted with VAMP2 proteins (v-vesicles) and encapsulating dual-labeled DNA hairpins are immobilized on the surface of the flow cell. Vesicles reconstituted with Syntaxin1A/SNAP-25 proteins (t-vesicles) and encapsulating unlabeled target DNA strands are flown in. (B) The time evolution of the radius of gyration (R_g) for Cy3/Cy5 dual-labeled DNA hairpin and unlabeled target DNA. The average radius of gyration R_g over the last 25-ns simulations for each DNA molecule shown in the black dash line is 1.91 ± 0.53 nm and 1.80 ± 0.28 nm for the labeled DNA hairpin and unlabeled target DNA, respectively. The R_g of target DNA collapses rather quickly from a relatively extended configuration as shown in Fig. S4. The results are consistent with the known high flexibility of single-stranded DNA (persistent length of 1.5 nm). (C) The snapshots of the equilibrated system for the unlabeled target DNA (Upper) and the dual-labeled DNA hairpin (Lower) resulting from 75-ns MD simulations.

Fig. 3.

Single-vesicle content mixing of DNA cargoes. (A) Typical time traces of fluorescence intensities (green curve for the donor and red curve for the acceptor) and the corresponding FRET efficiency (blue curve) for the DNA probes that show a content-mixing event. The red laser excitation was used to verify that the FRET change was induced by DNA annealing, not from photobleaching of the acceptor dye. The photobleaching probability was ∼0.4%. More time traces are shown in Fig. S6. Examples of photobleaching and leakage are shown in Fig. S7. (B) The cumulative fusion time histograms determined from 195 traces for large DNA probes was overlaid with that for sulforrhodamine B. Syt1 is present in all experimental runs. Blue arrows indicate the arrival of Ca²⁺. (Inset) The first 10 s of fusion time histograms. The sample size of docked vesicle pairs was 5,383. (C) Energy landscape for fusion pore dynamics. The activation free energy was calculated from a standard free energy equation k_exp/k_spo = exp(−ΔG^‡/RT), where k_exp is the rate constant for pore expansion and k_spo is that of small pore opening. (D) Content mixing for the small and large indicators during initial 60 s with or without Ca²⁺. The data were derived directly from three independent experiments for both small and large content indicators. The error bars, which represent SD, were obtained from three independent measurements.

Fig. 4.

Complexin 1 accelerates fusion pore expansion for Ca²⁺-triggered fusion mediated by SNARE and Syt1. Complexin 1 promotes fusion pore opening probed by small sulforhodamine B (A) only modestly, but it stimulates fusion pore expansion probed large DNA probes (B) significantly. Results were obtained by more than three independent experiments. (C) Bar graph analysis of content-mixing events at t = 60 s for both small and large cargos. The error bars, which represent SD, were obtained from three to six independent measurements. (D) Bar graph of large DNA probes for Ca²⁺-triggered fusion and in the presence of full-length or truncated (amino acid 41–134) complexin 1. The error bars, which represent SD, were obtained from three to six independent measurements.